Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/40—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum bacterial
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/08—Clostridium, e.g. Clostridium tetani
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
  • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Definitions

• C. difficile infection classified as an urgent public health threat by the Centers for Disease Control, is a bacterial toxin-mediated disease and a leading cause of hospital acquired infections.
• CDI C. difficile infection
• the majority of CDI is precipitated by intestinal microbiome dysbiosis (disruption of normal gut flora), a result of prior treatment with broad-spectrum antibiotics, which facilitates the proliferation of C. difficile.
• the dysbiosis which allows this pathogen to cause disease is prolonged by the very antibiotics used to treat CDI, resulting in a high rate of disease recurrence.
• Clostridium difficile a Gram-positive spore-forming anaerobe
• C. difficile spores are resistant to most disinfectants and are shed into the hospital environment by both symptomatic patients and asymptomatic carriers.
• the annual rate of CDI has doubled since 2001, coincident with the emergence of hypervirulent strains.
• Over 500,000 new cases of C. difficile infection occur each year in the US and estimates suggest greater than 400,000 diagnosed CDI events occur annually in Europe. This represents a substantial burden of morbidity, mortality, and healthcare resource consumption that calls for a more effective treatment strategy.
• CDI is most common in elderly patients with comorbidities— a fragile population— and infections are typically subsequent to treatment with broad-spectrum antibiotics.
• Antibiotic- mediated disruption of the beneficial intestinal microbiota allows colonization and infection with C. difficile.
• the antibiotics commonly used to treat CDI (metronidazole, vancomycin and fidaxomicin) prolong intestinal dysbiosis and lead to a 13-25% rate of infection recurrence following cessation of antibiotic therapy.
• a lasting cure for CDI requires the restoration of a diverse and protective intestinal microbiome that is resistant to infection recurrence. Indeed, it has been advances in understanding of C. difficile pathogenesis and resistance that have helped clarify the important role of the beneficial gut microbiome in maintaining overall health. At present, effective treatments and preventatives for C. difficile infection and illness are lacking. New methods of treatment are urgently required.
• the invention generally features methods for treating C. difficile infection (CDI), C. difficile associated disease, and symptoms thereof, featuring the use of antibodies having enhanced half-life that specifically bind C. difficile toxin A and/or toxin B.
• CDI C. difficile infection
• C. difficile associated disease C. difficile associated disease
• symptoms thereof featuring the use of antibodies having enhanced half-life that specifically bind C. difficile toxin A and/or toxin B.
• the invention provides a method of treating a C. difficile infection or C.
• ⁇ 3 ⁇ 4 zcz7e-associated disease in a subject the method involving administering to the subject a combination of an anti-C. difficile toxin A antibody and an anti-C. difficile toxin B antibody having an alteration that increases the half-life of one or both antibodies relative to anti-C.
• the invention provides a method of treating a C. difficile infection or C.
• ⁇ 3 ⁇ 4 zcz7e-associated disease in a subject the method involving administering to the subject a combination of an anti-C. difficile toxin A antibody and an anti-C. difficile toxin B antibody and vancomycin, to thereby reduce the dose or dose frequency of vancomycin relative to a reference dose or dose frequency.
• one or both antibodies have increased half-life relative to anti-C. difficile toxin A and B antibodies lacking the alteration.
• the alteration is any one or more of 252Y, 254T, or 256E (e.g., YTE modification).
• the alteration is conjugation to polyethylene glycol (PEG) or conjugation to albumin.
• the anti-toxin A antibody has a heavy chain containing the sequence SEQ ID NO: 1:
• the anti-toxin B antibody has a heavy chain containing the sequence SEQ ID NO: 3:
• the anti-toxin B antibody has a light chain containing the sequence SEQ ID NO: 4:
• the anti-toxin A antibody is PA50-YTE.
• the anti-toxin B antibody is PA41-YTE.
• the combination of the antibodies is PA50YTE/PA41 YTE COMBINATION.
• PA50YTE/PA41 YTE COMBINATION is administered in a single dose.
• the method of treatment further involves administering an antibiotic, such as vancomycin, fidaxomicin and metronidazole.
• an antibiotic such as vancomycin, fidaxomicin and metronidazole.
• the antibiotic is administered orally or intravenously.
• the method of treatment further involves administering vancomycin.
• the vancomycin is administered orally or intravenously.
• the reference dose and dose frequency is intravenous administration of vancomycin at 15-20 mg/kg, 2-3 times daily.
• the reference dose and dose frequency is oral administration at 125 mg, 3-4 times daily.
• C. difficile toxin A and/or toxin B are neutralized.
• the method of treatment reduces the time to C. difficile reinfection.
• the method of treatment enhances microbiome restoration, reduces microbiome dysbiosis, and/or reduces intestinal damage in the subject, including for example, relative to an antibiotic therapy.
• Clostridium difficile toxin A is meant a polypeptide or fragment thereof having at least about 85% or greater amino acid identity to the amino acid sequence provided at NCBI Accession No. YP_001087137 and having TcdA biological activity.
• TcdA biological activity includes glucosylating activity, such as glucosylation of GTPases (e.g., Rho, Rac, and Cdc42).
• GTPases e.g., Rho, Rac, and Cdc42.
• An exemplary C. difficile toxin A sequence is provided below (SEQ ID NO: 5): 1 msliskeeli klaysirpre neyktiltnl deynklttnn nenkylqlkk lnesidvfmn
• TcdB biological activity includes glucosylating activity, such as glucosylation of GTPases (e.g., Rho, Rac, and Cdc42).
• GTPases e.g., Rho, Rac, and Cdc42.
• An exemplary C. difficile toxin B sequence is provided below (SEQ ID NO: 6):
• half-life or "in vivo half-life” as used herein refers to a biological half-life of an antibody (e.g., IgG), or a fragment thereof, containing FcRn-binding sites in the circulation of a given animal and is represented by a time required for half the quantity administered in the animal to be cleared from the circulation and/or other tissues in the animal.
• an antibody e.g., IgG
• fragment thereof containing FcRn-binding sites in the circulation of a given animal and is represented by a time required for half the quantity administered in the animal to be cleared from the circulation and/or other tissues in the animal.
• the curve is usually biphasic with a rapid a-phase which represents an equilibration of the injected IgG molecules between the intra- and extra-vascular space and which is, in part, determined by the size of molecules, and a longer ⁇ -phase which represents the catabolism of the IgG molecules in the intravascular space.
• the term "in vivo half-life" practically corresponds to the half-life of the IgG molecules in the ⁇ - phase.
• antibody having increased half-life is meant an antibody having increased biological half-life when compared to a reference antibody.
• the reference antibody is an antibody that lacks an alteration or modification (e.g., an unmodified parent or precursor antibody).
• anti-tcdA antibody is meant an antibody that specifically binds C. difficile toxin A.
• Anti-tcdA antibodies include monoclonal and polyclonal antibodies that are specific for C. difficile toxin A, and antigen-binding fragments thereof.
• anti-tcdA antibodies as described herein are monoclonal antibodies (or antigen-binding fragments thereof), e.g., murine, humanized, or fully human monoclonal antibodies, including modified derivatives thereof.
• Exemplary anti-tcdA antibodies e.g., PA-50, PA-39, and PA-38
• PA50-YTE which has the following heavy and light chain sequences:
• PA50-YTE Light Chain (SEQ ID NO: 2):
• PA50-YTE Heavy Chain (SEQ ID NO: 1):
• anti-tcdB antibody an antibody that specifically binds C. difficile toxin B.
• Anti-tcdB antibodies include monoclonal and polyclonal antibodies that are specific for C.
• anti-tcdB antibodies as described herein are monoclonal antibodies (or antigen-binding fragments thereof), e.g., murine, humanized, or fully human monoclonal antibodies, including modified derivatives thereof.
• Exemplary anti-tcdB antibodies e.g., PA-41) are described in US20130202618 / US8986697, which are incorporated herein by reference in their entireties.
• the anti-tcdB antibody is PA41-YTE, which has the following heavy and light chain sequences: PA41-YTE Light Chain (SEQ ID NO: 4)
• PA41-YTE Heavy Chain (SEQ ID NO: 3)
• ameliorate decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
• antibody refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen- binding site, regardless of whether it is produced in vitro or in vivo.
• the term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single- chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies.
• antibody also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen -binding function, i.e., the ability to bind a C. difficile toxin A or toxin B polypeptide specifically. Typically, such fragments would comprise an antigen-binding domain.
• an antigen-binding domain refers to a part of an antibody molecule that comprises amino acids responsible for the specific binding between the antibody and the antigen. In instances, where an antigen is large, the antigen-binding domain may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding domain is referred to as “epitope” or "antigenic determinant.”
• an antigen-binding domain comprises an antibody light chain variable region (V L ) and an antibody heavy chain variable region (V H ), however, it does not necessarily have to comprise both. For example, a so- called Fd antibody fragment consists only of a V H domain, but still retains some antigen-binding function of the intact antibody.
• Binding fragments of an antibody are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain antibodies.
• An antibody other than a "bispecific” or “bifunctional” antibody is understood to have each of its binding sites identical. Digestion of antibodies with the enzyme, papain, results in two identical antigen-binding fragments, known also as "Fab” fragments, and a "Fc” fragment, having no antigen-binding activity but having the ability to crystallize.
• Fv when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites.
• Fab when used herein refers to a fragment of an antibody that comprises the constant domain of the light chain and the CHI domain of the heavy chain.
• mAb refers to monoclonal antibody.
• Antibodies of the invention comprise without limitation whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies.
• C. ⁇ 3 ⁇ 4 z ' cz7e-associated disease is meant any disease or symptom thereof associated with a C. difficile infection.
• C. ⁇ 3 ⁇ 4 z ' cz7e-associated diseases are characterized by one or more of the following symptoms: diarrhea, pseudomembranous colitis, toxic megacolon, perforation of the colon, and, in some instances, sepsis.
• the term "effective amount” refers to a dosage or amount of an agent that is sufficient to reduce or stabilize a C. difficile infection in a subject or to reduce and/or ameliorate symptoms associated with a C. difficile infection in a patient or to otherwise achieve a desired biological outcome.
• neutralize refers to the reduction, inhibition, blocking, amelioration, or elimination of adverse effect(s) of the toxin(s) which the antibody(ies) specifically bind.
• Neutralization of adverse effect(s) of the toxin(s) includes 1) delaying, reducing, inhibiting, or preventing onset or progression of C. difficile infection or C. dz7Jz ' cz7e-associated diarrhea or disease, 2) increasing survival of a subject as compared to the median survival of subjects who have not been treated with the antibody(ies) and who have C. difficile infection or C. difficile- associated disease, 3) eliminating one or more symptoms or adverse effects or reducing the severity of one or more symptoms or adverse effects associated with C. difficile infection or C. dz7Jz ' cz7e-associated diarrhea or disease, 4) allowing for the repopulation of the normal microflora of the gastrointestinal tract of subjects who are or have been infected with C.
• C. difficile 5) preventing a recurrence of C. difficile infection or C. dz7Jz ' cz7e-associated disease in subjects who have been afflicted with C. difficile infection or C. dz7Jz ' cz7e-associated disease, 6) effecting a cure rate of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% in subjects who have C. difficile infection or C. dz7Jz ' cz7e-associated disease, and/or 7) preventing death due to CD AD or other adverse events associated with C. difficile infection.
• isolated refers to a molecule that is substantially free of other elements present in its natural environment.
• an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived.
• isolated also refers to preparations where the isolated protein is sufficiently pure to be administered as a pharmaceutical composition, or at least 70-80% (w/w) pure, more preferably, at least 80-90% (w/w) pure, even more preferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.
• fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. In a particular embodiment, a fragment of a polypeptide may contain 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 300 amino acids.
• reference is meant a standard of comparison.
• a “reference sequence” is a defined sequence used as a basis for sequence comparison.
• a reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
• the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids.
• the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
• telomere binding an agent (e.g. , antibody) that recognizes and binds a molecule (e.g., polypeptide), but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample.
• an agent e.g. , antibody
• molecule e.g., polypeptide
• two molecules that specifically bind form a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity.
• binding is considered specific when the affinity constant KA IS higher than 10 7 M "1 , or more preferably higher than 10 s M _1 .
• subject is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, feline, or murine.
• the term "about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
• compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
• Figure 1 shows that PA50YTE/PA41 YTE COMBINATION, a combination of anti-toxin A and anti-toxin B monoclonal antibodies having enhanced half-life, provided a superior post infection protective benefit relative to antibiotic treatment in a C. difficile hamster infection model.
• a graph depicts the survival results of the groups of animals of the study. As depicted in the schematic, animals were challenged with C. difficile spores orally at day 0 of the study and were treated with clindamycin (10 mg/kg) at day 1. Study groups included infected control animals receiving no treatment, animals treated with vancomycin, and animals treated with a combination of murine anti-toxin A and anti-toxin B monoclonal antibodies having enhanced half-life. Animals treated with a combination of the anti-toxin A and anti-toxin B monoclonal antibodies survived and were protected against C. difficile toxicity for the duration of the study.
• the invention features methods for treating C. difficile infection (CDI), C. difficile associated disease, and symptoms thereof, featuring antibodies having enhanced half-life that specifically bind C. difficile toxin A and/or toxin B.
• CDI C. difficile infection
• C. difficile associated disease C. difficile associated disease
• symptoms thereof featuring antibodies having enhanced half-life that specifically bind C. difficile toxin A and/or toxin B.
• the present invention is based, at least in part, on the discovery that a mixture of two monoclonal antibodies (mAbs) having increased half-life, neutralizes C. difficile toxins A and B, the key virulence factors of this pathogen.
• This combination represents a pathogen-focused, precision medicine alternative to antibiotic therapy.
• toxin neutralization by such a combination was at least as effective, if not more effective, than antibiotics in treating CDI.
• CDI C. difficile infection
• CDAD C. difficile -associated disease
• C. ⁇ 3 ⁇ 4 z ' cz7e-associated disease typically is precipitated by the disruption of the colonic flora through the use of antibiotics such as clindamycin, cephalosporins, and
• a benefit can be the elimination of one or more symptoms or adverse effects, or a reduction in, or amelioration of, the severity of the one or more symptoms or adverse effects that result from the infection or disease; a delay, halt, or reversal in the progression of the infection or disease; a recolonization, resurgence, or repopulation of the normal and natural microflora of the gastrointestinal tract, colon, bowel, etc., or the cure of the infection or disease (i.e., a clinician would evaluate the subject and determine that the subject no longer has the infection or disease).
• Symptoms or adverse effects associated with C. difficile infection include dehydration, diarrhea, cramping, kidney failure, bowel perforation, toxic megacolon, which can lead to rupture of the colon, and death.
• the therapeutic methods provided can be used to reduce, diminish, ameliorate, or eliminate any or all of the symptoms or adverse effects provided herein.
• C. difficile infection refers to an infection that results from the presence of C. difficile in the intestinal flora where it was not previously present or a change in the presence of C. difficile in the intestinal flora (e.g., an increase in the total amount of C.
• toxins A and B demonstrate different pathological profiles with potential synergy in causing disease.
• toxin A is an enterotoxin that induces diarrhea, while toxin B does not elicit a fluid response in this species.
• toxin B is more potently cytotoxic in vitro.
• C. difficile infection commonly manifests as mild-to-moderate diarrhea, occasionally with abdominal cramping. Pseudomembranes, which are adherent yellowish-white plaques on the intestinal mucosa, are occasionally observed.
• patients with C. difficile infection can present with an acute abdomen and fulminant life-threatening colitis, which results from a disruption of the normal bacterial flora of the colon, colonization with C. difficile and release of toxins that cause mucosal inflammation and damage.
• Antibiotic therapy is the key factor that alters the colonic flora. While normal gut flora resists colonization and overgrowth with C. difficile, antibiotic use, which suppresses the normal flora, allows C. difficile bacteria to proliferate.
• the mAbs of the present invention are utilized for the treatment of subjects who are asymptomatic, but who are susceptible to, or at risk of, contracting C. difficile infection and becoming afflicted with its associated diseases. Such subjects may be hospitalized or may be outside of a hospital setting.
• C. ⁇ 3 ⁇ 4 z ' cz7e-associated disease is prior exposure to antibiotics.
• the most common antibiotics implicated in C. difficile colitis include cephalosporins (especially second and third generation), ampicillin/amoxicillin and clindamycin. Less commonly implicated antibiotics are the macrolides (i.e., erythromycin, clarithromycin, azithromycin) and other penicillins.
• Compounds or other agents which are occasionally reported to cause the disease include aminoglycosides, fluoroquinolones, trimethoprim- sulfamethoxazole,
• C. difficile colitis Even brief exposure to any single antibiotic can cause C. difficile colitis, particularly if normal intestinal flora are adversely affected or killed. A prolonged antibiotic course, or the use of two or more antibiotics, increases the risk of disease. Antibiotics traditionally used to treat C. difficile colitis have been shown to cause disease. Other risk factors associated with infection by C.
• C. difficile include advanced age (>65 years); weakened immune system; recent hospitalization (particularly sharing a hospital room with an infected patient, intensive care unit stays and prolonged hospital stays); living in a nursing home, hospice, or other longterm care facility; abdominal surgery; chronic colon disease, (e.g., inflammatory bowel disease (IBD) or colorectal cancer); taking prescription or over the counter antacids which may reduce stomach acid and allow C. difficile to pass more easily into the intestine; and a previous C. difficile infection. More factors associated with C. difficile disease include antineoplastic agents, principally methotrexate, hemolytic-uremic syndrome, malignancies, intestinal ischemia, renal failure, necrotizing enterocolitis,
• the subjects that can be administered the therapies provided herein include any of the subjects described that are at risk for C. difficile infection.
• C. ⁇ 3 ⁇ 4 z ' cz7e-associated diarrhea can be a serious condition with a mortality rate of up to 25% in elderly patients who are frail. Reports that focus on more seriously ill patients indicate mortality rates of 10-30%.
• C. difficile infection is more common in elderly people, and old age may promote susceptibility to colonization and disease. While infants and young children frequently harbor C. difficile and its toxins, clinical infection is uncommon. Cross-infection by C. difficile is common in neonatal units, but neonates do not seem to develop C. ⁇ 3 ⁇ 4 z ' cz7e-associated diarrhea.
• the disclosure provides methods of treating C. difficile infection, C. zYJz ' cz ' /e-associated disease, and symptoms thereof, comprising the use of one or more isolated antibodies having enhanced half-life, or antigen-binding fragments thereof, which inhibit, block, or prevent C. difficile toxin A and/or toxin B toxicity or activity.
• C. difficile pathology is driven by two secreted toxins, A and B, which mediate the colitis, diarrhea and massive inflammatory response characteristic of this disease.
• Toxins A and B are the major virulence determinants of C.
• Toxins A and B are transcribed from a pathogenicity locus that includes the toxin genes, tcdA (toxin A) and tcdB (toxin B), and three regulatory genes, one of which (tcdC) encodes a putative negative regulator of toxin
• TcdC protein appears to inhibit toxin transcription during the early, exponential- growth phase of the bacterial life cycle.
• toxin B an autocatalytic cleavage site between leucine543 and glycine544 has been described. Cleavage results from activation of an aspartyl protease domain by host cytosolic inositol phosphate, and releases the active glucosyltransferase domain.
• PA50YTE/PA41 YTE COMBINATION is an equimolar mixture of two fully human monoclonal antibodies having enhanced half-life which bind to and neutralize the cytotoxicity of toxins A and B.
• COMBINATION demonstrated greater toxin neutralizing potency in vitro and neutralized toxins from a broader range of clinical isolates.
• COMBINATION demonstrated greater toxin neutralizing potency in vitro and neutralized toxins from a broader range of clinical isolates.
• PA50YTE/PA41YTE COMBINATION provided superior protection when compared to existing antitoxin monoclonal antibodies.
• PA50YTE/PA41 YTE COMBINATION are engineered with extended half-life technology providing a 3-fold expanded window of toxin neutralization compared to standard IgG, providing months of prophylaxis against infection recurrence.
• Treatment with anti-toxin A and anti-toxin B antibodies having enhanced half-life can allow for the restoration of normal gut flora in a subject infected with C. difficile. Such antibodies can resolve disease in patients undergoing treatment.
• Treatment with anti-toxin A and anti-toxin B antibodies having enhanced half-life can also demonstrate beneficial in vivo pharmacokinetics. Treatment with anti-toxin A and anti-toxin B antibodies having enhanced half-life can also provide prolonged or long lasting therapy for a subject who has been infected with C. difficile.
• long lasting refers to therapy that results in an absence of C. difficile infection or C. ⁇ 3 ⁇ 4 z ' cz7e-associated disease one month or more after cessation of treatment.
• the therapy results in an absence of C. difficile infection or C. ⁇ 3 ⁇ 4 zcz7e-associated disease for two or more months.
• therapy with mAbs of the invention results in treating or depressing active C. difficile infection and in reducing or diminishing the robustness of infection.
• therapy provided by the invention results in an absence of C. difficile infection or C. dz7Jz ' cz7e-associated disease in a subject for 1, 2, 3, 4, 5, or 6 months.
• therapy provided by the invention results in an absence of C. difficile infection or C. dz7Jz ' cz7e-associated disease in a subject for longer than 6 months.
• Treatment with anti-toxin A and anti-toxin B antibodies having enhanced half-life can prevent recurrence of C. difficile infection and/or C. dz7Jz ' cz7e-associated disease.
• treatment with anti-toxin A and anti-toxin B antibodies having enhanced half-life can effect a cure or survival rate of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%.
• the antibodies can effect a cure or survival rate of 100%.
• one or more anti-toxin A antibodies when administered to a subject, together with one or more anti-toxin B antibodies, effect a cure or survival rate of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%.
• cure rate refers to the percentage of subjects that a clinician would determine to no longer have the infection or disease out of a population of subjects with the infection or disease administered one or more antibodies, or one or more therapeutic methods thereof, of the invention.
• Sudvival rate refers to the percentage of subjects that survive for a desired period of time out of a population of subjects administered one or more antibodies, or one or more therapeutic methods thereof, of the invention.
• PA50YTE/PA41 YTE COMBINATION provides a continuous window of toxin neutralization further minimizing the recurrence of CDI.
• PA50YTE/PA41 YTE COMBINATION is an example of a precision medicine that effectively treats a difficult bacterial infection without the collateral damage to the beneficial microbiome associated with traditional antibiotic therapy.
• PA50YTE/PA41 YTE COMBINATION is at least as effective as vancomycin in treating C. difficile infections.
• COMBINATION acts by competitively inhibiting toxin binding to the intestinal wall, thereby rendering the wall less susceptible to C. difficile infection.
• vancomycin is a bactericidal agent.
• vancomycin and PA50YTE/PA41YTE COMBINATION may be administered concurrently. Such combined therapeutic strategy would likely require a lower dose or reduced frequency of administration of vancomycin than conventional vancomycin therapy, thereby reducing adverse side effects, enhancing microbiome restoration, reducing microbiome dysbiosis, and/or reducing the risk of re-infection.
• Vancomycin dosages are calculated on actual body weight (ABW). However, for obese patients, initial dosing is based on ABW and then adjusted based on serum vancomycin concentrations to achieve therapeutic levels. Vancomycin dosages of 15-20 mg/kg (based on ABW) given every 8-12 hours achieve target serum concentrations of MIC ⁇ 1 mg/L in most patients with normal renal function (e.g., 1 g every 12 hours). In one embodiment, a maintenance dose (about 15-20 mg/kg of actual body weight, rounded to the nearest 250 mg) is administered at the dosing interval recommended for a patient's creatinine clearance levels
• Vancomycin Maintenance doses and infusion rates are provided at Table 1. Table 1. Vancomycin Maintenance Doses and Infusion Rates
• a loading dose of 25-30 mg/kg (based on ABW) can be used.
• a one-time loading dose of about 25-30 mg/kg of actual body weight (rounded to the nearest 250 mg) at a rate of about 500 mg/hour (but no more than about 1 g/hr) may be considered for seriously ill patients (e.g., sepsis, fever and neutropenia, suspected/proven MRSA bacteremia) with CrCL > 30 mL/min to rapidly attain therapeutic concentrations.
• Exemplary vancomycin loading doses and infusion rates are provided at Table 2.
• Vancomycin should be administered intravenously over an infusion period of at least 1 hour to minimize infusion related adverse effects. Vancomycin may be administered by intermittent dosing or continuous infusion. When individual doses exceed 1 g (i.e., 1.5 and 2 g), the infusion period should be extended to 1.5-2 hours. Vancomycin dosing intervals are based in part on a patient's creatinine clearance levels (CrCL). For example, vancomycin dosing intervals based on estimated CrCL are provided at Table 3.
• vancomycin may be administered orally to reach the site of infection in the colon.
• a conventional regimen is vancomycin administered orally at about 125 mg about every 6 hr for 10 days.
• a conventional regimen is vancomycin administered orally at about 40 mg/kg/day about every 6-8 hours for 7-10 days; not to exceed 2 g/day.
• the fecal concentration of vancomycin may be about 500 ⁇ g/ml (Edlund et al., Clinical Infectious Diseases, 1997; 25 (3): 729-32) compared to MIC ⁇ 2 ⁇ g/ml for sensitive strains of C. difficile (Pelaez et al., Antimicrob Agents Chemother, 2002; 46 (6): 1647-1650).
• Trough serum vancomycin concentrations are the most accurate and practical method for monitoring vancomycin effectiveness. Trough concentrations should be obtained just before the next dose at steady state conditions. Steady-state achievement is variable and dependent on multiple factors. Trough samples should be obtained just before the fourth dose in patients with normal renal function to ensure that target concentrations are attained. Based on the potential to improve penetration, increase the probability of optimal target serum vancomycin
• Trough serum vancomycin concentrations in that range should achieve an AUC (area under the concentration-versus-time curve)/MIC
• AUC/MIC ratio of >400 has been advocated as a target to achieve clinical effectiveness with vancomycin.
• Animal studies and limited human data appear to demonstrate that vancomycin is not concentration dependent and that the AUC/MIC is a predictive pharmacokinetic parameter for vancomycin. Based on evidence suggesting that exposure to trough serum vancomycin concentrations of ⁇ 10 mg/L can produce strains with resistance, it is recommended that trough serum vancomycin concentrations always be maintained above 10 mg/L to avoid development of resistance.
• a targeted AUC/MIC of >400 is not achievable with conventional dosing methods if the vancomycin MIC is >2 mg/L in a patient with normal renal function (i.e., CrCL of 70-100 mL/min). Therefore, alternative therapies should be considered.
• Vancomycin has long been considered a nephrotoxic and ototoxic agent.
• a patient should be identified as having experienced vancomycin-induced nephrotoxicity if multiple (at least two or three consecutive) high serum creatinine concentrations (increase of 0.5 mg/dL or >50% increase from baseline, whichever is greater) are documented after several days of vancomycin therapy in the absence of an alternative explanation.
• trough serum vancomycin concentrations to reduce nephrotoxicity is best suited to patients receiving aggressive dosing targeted to produce sustained trough drug concentrations of 15-20 mg/L or who are at high risk of toxicity, such as patients receiving concurrent nephrotoxins.
• obtaining once- weekly trough concentrations in hemodynamically stable patients is recommended.
• Patients receiving prolonged courses of vancomycin should have at least one steady- state trough concentration obtained (just before the fourth dose).
• Monitoring is also recommended for patients with unstable renal function (either deteriorating or significantly improving) and those receiving prolonged courses of therapy (over three to five days). Frequent (in some instances daily) trough concentration monitoring is advisable to prevent toxicity in hemodynamically unstable patients. The exact frequency of monitoring is often a matter of clinical judgment.
• the therapeutic methods described herein comprise the use of one or more isolated antibodies having enhanced half-life, including antigen-binding fragments and modified derivatives thereof, which inhibit, block, or prevent C. difficile toxin A and/or toxin B toxicity or activity.
• Exemplary anti-tcdA e.g., PA-50, PA-39, and PA-38
• anti-tcdB antibodies e.g., PA-41
• Exemplary antibodies may also comprise one or more of the VH, VL, heavy chain, and light chain sequences at SEQ ID NOs: 7-22.
• the invention provides methods of treatment comprising the use of an isolated antibody, or antigen -binding fragment thereof, which inhibits, blocks, or prevents toxin A internalization and cytocellular toxicity.
• the antibody is a monoclonal antibody.
• the antibody is a humanized or chimeric antibody.
• the antibody is PA-50 (ATCC Accession No. PTA-964) or humanized PA-50.
• the antibody is PA-39 (ATCC Accession No. PTA-9692) or humanized PA-39.
• the antibody binds toxin A outside of the receptor binding domain of toxin A of C. difficile.
• the methods comprise the use of isolated antibody, or antigen-binding fragment thereof, which inhibits, blocks, or prevents C. difficile toxin B toxicity by binding to an epitopic site in the N-terminal enzymatic region of toxin B.
• the antibody is a monoclonal antibody.
• the antibody is a humanized or chimeric antibody.
• the antibody is PA-41 (ATCC Accession No. PTA- 9693) or a humanized form of PA-41.
• the antibody binds to the N- terminal enzymatic region of toxin B of C. difficile.
• the antibodies of the invention exhibit a number of beneficial characteristics.
• the anti-toxin A antibodies neutralize or inhibit the toxicity of toxin A both in vitro and in vivo.
• humanized PA-39 and humanized PA-41 demonstrated neutralization potencies (i.e., EC50 values; US20130202618 / US8986697) higher than those compared with values for neutralization by other human anti-toxin A and anti-toxin B
• the invention provides treatment with antibodies having enhanced half-lives.
• Anti-C. difficile toxin antibodies e.g., PA-39, PA-41, PA-50
• another functional molecule e.g., another peptide or protein (e.g., albumin).
• the antibodies can be linked by chemical cross-linking or by recombinant methods.
• the antibodies may also be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.
• the antibodies can be chemically modified by covalent conjugation to a polymer, for example, to increase their circulating half- life.
• exemplary polymers and methods to attach them are also shown in U.S. Pat. Nos.
• the Fc region of the antibody comprises at least one non- naturally occurring amino acid at one or more positions chosen from 252, 254, and 256.
• the non-naturally occurring amino acids are selected from the group chosen from 252Y, 254T and 256E (referred to as the "YTE modification"), as described in Dall'Acqua et al., J. Biol. Chem., 281, 23514-23524 (2006), and in US7083784 / US20030190311, each of which is incorporated herein by reference in their entireties.
• Antibodies having the YTE modification have enhanced half-lives compared to the unmodified antibodies (e.g., the parent antibody).
• PA-50-YTE is a fully human monoclonal antibody having enhanced half-life which binds to and neutralizes the cytotoxicity of toxin A.
• PA-41-YTE is a fully human monoclonal antibody having enhanced half-life which binds to and neutralizes the cytotoxicity of toxin B.
• the invention features a composition comprising an equimolar mixture of the anti-toxin A antibody PA-50-YTE and antitoxin B antibody PA-41-YTE termed PA50YTE/PA41 YTE COMBINATION (also termed PA50YTE/PA40YTE COMBINATION in priority application US 62/147,908 filed on
• an anti-toxin A antibody neutralizes or inhibits the in vivo toxicity of C. difficile toxin A at an effective dose.
• the anti-toxin B antibodies neutralize or inhibit the in vivo toxicity of toxin B.
• an effective dose of one or more anti-toxin A antibodies is provided to a C. difficile-infected subject.
• an effective dose of one or more anti-toxin A antibodies of the invention is provided in combination with an effective dose of one or more anti-toxin B antibodies of the invention to a C. difficile -infected subject.
• an anti-toxin A antibody of the invention in a 1: 1 combination with an anti-toxin B antibody of the invention is provided as an effective dose to a C. difficile-infected subject.
• an effective dose of an anti-toxin A antibody and an anti-toxin B antibody of the invention may be, for example, a 1 ⁇ 2: 1, 1: 1, 2: 1, 3: 1, 4: 1, etc., combination of the antibodies provided to a C. difficile-infected subject.
• the antibodies are humanized.
• the antibodies are included in a composition.
• an effective dose of the anti-toxin A and/or anti-toxin B antibodies may range from 0.1 ⁇ g to 1000 milligrams (mg).
• the anti-toxin A antibodies and anti-toxin B antibodies or antigen-binding fragments thereof may be administered to a subject in an amount of, for example, 0.1 mg/kg-150 mg/kg; in an amount of 0.5 mg/kg-75 mg/kg; in an amount of 1 mg/kg- 100 mg/kg; in an amount of 1 mg/kg-50 mg/kg; in an amount of 2 mg/kg-40 mg/kg; in an amount of 2 mg/kg-50 mg/kg; in an amount of 5 mg/kg-50 mg/kg; in an amount of 5 mg/kg-25 mg/kg; in an amount of 10 mg/kg-40 mg/kg; in an amount of 10 mg/kg-50 mg/kg; in an amount of 10 mg/kg-25 mg/kg; or in an amount of 15 mg/kg-50 mg/kg.
• the aforementioned amounts may comprise the varying ratios of anti-toxin A antibody and anti-toxin B antibody provided in combination.
• the dose or amount of the one or more anti-toxin A or anti-toxin B antibodies may range for example from 0.2 ⁇ g-250 ⁇ g, or from 2 ⁇ g-50 ⁇ g, or from 5 ⁇ g-50 ⁇ g, e.g., based on in vivo mouse studies.
• the dose or amount of one or more anti-toxin A or anti-toxin B antibodies may range for example from 2 mg/kg-40 mg/kg, 2 mg/kg- 50 mg/kg, 5 mg/kg-40 mg/kg, 5 mg/kg-50 mg/kg, 10 mg/kg-40 mg/kg, or 10 mg/kg-50 mg/kg, e.g., based on in vivo hamster studies.
• Antibodies provided herein include monoclonal antibodies produced by hybridomas that were deposited and given the following Patent Deposit Designations: PTA-9692 (for PA-39), PTA-9693 (for PA-41), PTA-9694 (for PA-50), and PTA-9888 (for PA-38). These hybridomas were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (“ATCC”), P.O. Box 1549, Manassas, Va. 20108 USA, as an International Depository Authority, on Jan. 6, 2009 (for PTA-9692, PTA-9693, PTA- 9694) and on Mar.
• ATCC American Type Culture Collection
• both the deposited hybridomas and the monoclonal antibodies produced by the hybridomas may be referred to by the same ATCC Deposit Designations or to the numbers found within the ATCC Deposit Designations.
• PTA-9888 or 9888 may be used to refer to the deposited hybridoma or to the monoclonal antibody produced by the hybridoma.
• the names of the monoclonal antibodies described herein may be used interchangeably with the names of the hybridomas that produce them. It will be clear to one of skill in the art when the name is intended to refer to the antibody or to the hybridoma that produces the antibody.
• the antigen-binding fragments provided herein include the antigen- binding fragments of the aforementioned deposited antibodies.
• Antibodies can be made, for example, using traditional hybridoma techniques (Kohler and Milstein (1975) Nature, 256: 495-499), recombinant DNA methods (U.S. Pat. No.
• Intact antibodies also known as immunoglobulins, are typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, designated as the ⁇ chain and the K chain, are found in antibodies.
• immunoglobulins can be assigned to five major classes: A, D, E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi, and IgA 2 .
• each light chain is composed of an N-terminal variable domain (V L ) and a constant domain (CL).
• Each heavy chain is composed of an N-terminal variable domain (VH), three or four constant domains (C H ), and a hinge region.
• the C H domain most proximal to V H IS designated as C H I.
• the VH and V L domains consist of four regions of relatively conserved sequence called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequence called complementarity determining regions (CDRs).
• the CDRs contain most of the residues responsible for specific interactions with the antigen.
• the three CDRs are referred to as CDR1, CDR2, and CDR3.
• CDR constituents on the heavy chain are referred to as HI, H2, and H3, while CDR constituents on the light chain are referred to as LI, L2, and L3, accordingly.
• CDR3 and, particularly H3, are the greatest source of molecular diversity within the antigen-binding domain.
• H3, for example, can be as short as two amino acid residues or greater than 26.
• a heavy chain CDR3 (H3) comprises between about 4 amino acids (see, for example, Ab No. 2) and 22 amino acids (see, for example, Ab Nos. 20 and 34).
• the Fab fragment (Fragment antigen-binding) consists of the VH-CH1 and VL-CL domains covalently linked by a disulfide bond between the constant regions.
• a so-called single chain (sc) Fv fragment (scFv) can be constructed.
• a scFv a flexible and adequately long polypeptide links either the C-terminus of the V H to the N-terminus of the VL or the C-terminus of the VL to the N-terminus of the VH.
• a 15-residue (Gly 4 Ser)3 peptide is used as a linker, but other linkers are also known in the art.
• Antibody diversity is a result of combinatorial assembly of multiple germline genes encoding variable regions and a variety of somatic events.
• the somatic events include recombination of variable gene segments with diversity (D) and joining (J) gene segments to make a complete VH region and the recombination of variable and joining gene segments to make a complete VL region.
• D diversity
• J joining
• the recombination process itself is imprecise, resulting in the loss or addition of amino acids at the V(D)J junctions.
• the structure for carrying a CDR will generally be an antibody heavy or light chain or a portion thereof, in which the CDR is located at a location corresponding to the CDR of naturally occurring VH and VL.
• the structures and locations of immunoglobulin variable domains may be determined, for example, as described in Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md., 1991.
• Anti-C. difficile toxin A and toxin B antibodies may optionally comprise antibody constant regions or parts thereof.
• a VL domain may have attached, at its C terminus, antibody light chain constant domains including human CK or C chains.
• a specific antigen -binding domain based on a VH domain may have attached all or part of an immunoglobulin heavy chain derived from any antibody isotope, e.g., IgG, IgA, IgE, and IgM and any of the isotope sub-classes, which include but are not limited to, IgGi and IgG 4 .
• V H and/or V L domains comprise a V H and/or V L domain of an Fv fragment from a C. difficile toxin A or toxin B antibody. Further embodiments comprise at least one CDR of any of these V H and V L domains.
• the V H and/or V L domains may be germlined, i.e., the framework regions (FRs) of these domains are mutated using conventional molecular biology techniques to match those produced by the germline cells. In other embodiments, the framework sequences remain diverged from the consensus germline sequences.
• the antibodies of this invention may be used to inhibit proteins that differ somewhat from toxin A or toxin B.
• the antibodies are expected to retain the specificity of binding so long as the target protein comprises a sequence which is at least about 60%, 70%, 80%, 90%, 95%, or more identical to any sequence of at least 100, 80, 60, 40, or 20 of contiguous amino acids of toxin A or toxin B.
• the percent identity is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altshul et al. (1990) J. Mol. Biol., 215: 403-410, the algorithm of Needleman et al. (1970) J. Mol. Biol., 48: 444-453, or the algorithm of Meyers et al. (1988) Comput. Appl. Biosci., 4: 11-17.
• BLAST Basic Local Alignment Tool
• epitope mapping see, e.g., Epitope Mapping Protocols, ed. Morris, Humana Press, 1996) and secondary and tertiary structure analyses can be carried out to identify specific 3D structures assumed by the disclosed antibodies and their complexes with antigens.
• Such methods include, but are not limited to, X-ray crystallography (Engstom (1974) Biochem. Exp. Biol., 11:7-13) and computer modeling of virtual representations of the presently disclosed antibodies (Fletterick et al. (1986) Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
• kits for treating a C. difficile infection or symptoms thereof includes a therapeutic composition containing an effective amount of one or more of an anti-toxin A antibody and/or anti-toxin B antibody having enhanced half-life in unit dosage form.
• the kit comprises a sterile container which contains a therapeutic or prophylactic biological composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
• Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
• an antibody of the invention is provided together with instructions for administering the antibody or agent to a subject having or at risk of developing C. difficile infection, C. difficile associated disease, or symptoms thereof.
• the instructions will generally include information about the use of the antibodies for the treatment or prevention of such indications.
• the instructions include at least one of the following:
• the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
• Example 1 Treatment with a combination of anti-toxin A and anti-toxin B monoclonal antibodies increased survival and protected against toxicity in a model of C. difficile infection.
• C. difficile infection reproduces key aspects of C. difficile- Associated Diarrhea (CDAD) disease in humans.
• CDAD C. difficile- Associated Diarrhea
• Hamsters were challenged with C. difficile spores by oral administration at day 0 and pretreated with a single dose of clindamycin (10 mg/kg) at day 1 to disrupt the normal colonic flora. Animals were placed in a control group receiving no treatment and groups receiving either vancomycin (on days 2, 3, 4, 5, and 6) or a combination of toxin A and toxin B antibodies PA- 50-YTE (40 mg/kg) and PA-41-YTE (40 mg/kg), also termed MEDI095, on day 2. Animals were monitored daily for health status and survival.
• SEQ ID NO: 8 Anti-toxin A antibody, VH region of a humanized PA- 39 (hPA-39) Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
• SEQ ID NO: 14 Anti-toxin B antibody, VH region of a humanized PA-41 (hPA-41) Gin Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
• Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val
• SEQ ID NO: 18 Anti-toxin A antibody, light chain Met Asp Phe Gin Val Gin He Phe Ser Phe Leu Leu He Ser Ala Ser 1 5 10 15
• Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys
• Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val

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